Method of providing safe administration of adenoviral vectors encoding a zika virus antigen

ABSTRACT

Provided are methods for generating an immune response against a Zika virus in a human subject in need thereof. The methods comprise administering to the subject a pharmaceutical composition comprising adenoviral vectors encoding a Zika virus antigen and a pharmaceutically acceptable carrier.

FIELD OF THE INVENTION

This invention relates to biotechnology. More particularly, to methodsof administering adenovirus viral particles comprising an optimized,non-naturally occurring Zika virus (ZIKV) nucleic acid molecule to asubject in need thereof for preventing a ZIKV infection and/or symptomscaused by a ZIKV.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. patent application Ser. No.16/194,868, filed Nov. 19, 2018, which claims priority to U.S.Provisional Application No. 62/588,635, filed Nov. 20, 2017. Eachdisclosure is incorporated herein by reference in its entirety.

REFERENCE TO SEQUENCE LISTING SUBMITTED ELECTRONICALLY

This application contains a sequence listing, which is submittedelectronically via EFS-Web as an ASCII formatted sequence listing with afile name “004852_11773_Sequence_Listing” and a creation date of Nov.10, 2020, and having a size of 186 kb. The sequence listing submittedvia EFS-Web is part of the specification and is herein incorporated byreference in its entirety.

BACKGROUND OF THE INVENTION

Zika virus (ZIKV) is a flavivirus that is responsible for anunprecedented current epidemic in Brazil and the Americas. ZIKV has beencausally associated with microcephaly, intrauterine growth restriction,and other birth defects in humans and in murine models. ZIKV is believedto cause neuropathology in developing fetuses by crossing the placentaand targeting cortical neural progenitor cells, leading to impairedneurogenesis and resulting in microcephaly and other congenitalmalformations.

The World Health Organization declared the clusters of microcephaly andneurological disorders and their association with ZIKV infection to be aglobal public health emergency on Feb. 1, 2016. ZIKV also has beenassociated with neurologic conditions such as Guillain-Barré syndrome.While the rapid development of a safe and effective ZIKV vaccine is aglobal health priority, very little is currently known about ZIKVimmunology and mechanisms of immune protection.

Accordingly, there is an unmet need in the field for ZIKV vaccines.

BRIEF SUMMARY OF THE INVENTION

Provided herein are methods for generating an immune response against aZika virus in a human subject in need thereof. A method according to anembodiment of the invention comprises administering to the subject apharmaceutical composition comprising adenoviral vectors comprising anucleic acid sequence encoding a Zika virus antigen and apharmaceutically acceptable carrier, wherein about 1×10¹⁰ adenoviralvectors to about 5×10¹¹ adenoviral vectors, preferably about 5×10¹⁰adenoviral vectors to about 1×10¹¹ adenoviral vectors are administeredto the subject in need thereof. In a preferred embodiment, about 5×10¹⁰adenoviral vectors are administered per dose to the human subject inneed thereof. In another preferred embodiment, about 1×10¹¹ adenoviralvectors are administered per dose to the human subject in need thereof.

In certain embodiments, the pharmaceutical composition is administeredvia an intramuscular injection to the human subject in need thereof. Incertain embodiments, the pharmaceutical composition is administered tothe human subject as a single dose. In certain embodiments, thepharmaceutical composition is administered to the human subject as adouble dose. The first and second dose of the pharmaceutical compositioncan be administered to the human subject about four weeks, about eightweeks, about twelve weeks, about three months, about six months, aboutnine months, about one year, or about two years apart. In certainembodiments, the first and second dose of the pharmaceutical compositioncan be administered to the human subject about eight weeks apart.

In certain embodiments, the Zika virus antigen comprises the amino acidsequence of SEQ ID NO:2, 4, 6, 8, 10, or 12. In certain embodiments, theZika virus antigen comprises the amino acid sequence of SEQ ID NO:2.

In certain embodiments, the adenoviral vectors are of the Ad26 serotype.

BRIEF DESCRIPTION OF THE DRAWINGS

The foregoing summary, as well as the following detailed description ofthe invention, will be better understood when read in conjunction withthe appended drawings. It should be understood that the invention is notlimited to the precise embodiments shown in the drawings.

FIGS. 1A and 1B show the ZIKV neutralizing antibody response (assessedby MN50 VNA) in serum from clinical study participants at the indicatedstudy timepoints (days 15 and 29 on FIG. 1A and days 57, 71 and 85 onFIG. 1B) for each of the tested regimens. Doses were given at days 1 and57. Responders are depicted on FIGS. 1A and 1B with filled circles,non-responders are depicted with open light grey circles. Pooledbaseline is depicted with dark grey circles on FIG. 1A on the left-handside of the graph. LD stands for Low Dose which stands for a dose of5×10¹⁰ viral particles (vp) of Ad26.ZIKV.001; HD stands for High Dose,which stands for a dose of 1×10¹¹ vp Ad26.ZIKV.001, PL stands forPlacebo.

DETAILED DESCRIPTION OF THE INVENTION

Various publications, articles and patents are cited or described in thebackground and throughout the specification; each of these references isherein incorporated by reference in its entirety. Discussion ofdocuments, acts, materials, devices, articles or the like which has beenincluded in the present specification is for the purpose of providingcontext for the invention. Such discussion is not an admission that anyor all of these matters form part of the prior art with respect to anyinventions disclosed or claimed.

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood to one of ordinary skill inthe art to which this invention pertains. Otherwise, certain terms usedherein have the meanings as set forth in the specification.

It must be noted that as used herein and in the appended claims, thesingular forms “a,” “an,” and “the” include plural reference unless thecontext clearly dictates otherwise.

Unless otherwise stated, any numerical values, such as a concentrationor a concentration range described herein, are to be understood as beingmodified in all instances by the term “about.” Thus, a numerical valuetypically includes ±10% of the recited value. For example, aconcentration of 1 mg/mL includes 0.9 mg/mL to 1.1 mg/mL. Likewise, aconcentration range of 1% to 10% (w/v) includes 0.9% (w/v) to 11% (w/v).As used herein, the use of a numerical range expressly includes allpossible subranges, all individual numerical values within that range,including integers within such ranges and fractions of the values unlessthe context clearly indicates otherwise.

Unless otherwise indicated, the term “at least” preceding a series ofelements is to be understood to refer to every element in the series.Those skilled in the art will recognize or be able to ascertain using nomore than routine experimentation, many equivalents to the specificembodiments of the invention described herein. Such equivalents areintended to be encompassed by the invention.

It should also be understood that the terms “about,” “approximately,”“generally,” “substantially” and like terms, used herein when referringto a dimension or characteristic of a component of the preferredinvention, indicate that the described dimension/characteristic is not astrict boundary or parameter and does not exclude minor variationstherefrom that are functionally the same or similar, as would beunderstood by one having ordinary skill in the art. At a minimum, suchreferences that include a numerical parameter would include variationsthat, using mathematical and industrial principles accepted in the art(e.g., rounding, measurement or other systematic errors, manufacturingtolerances, etc.), would not vary the least significant digit.

As used herein, the terms “comprises,” “comprising,” “includes,”“including,” “has,” “having,” “contains” or “containing,” or any othervariation thereof, will be understood to imply the inclusion of a statedinteger or group of integers but not the exclusion of any other integeror group of integers and are intended to be non-exclusive or open-ended.For example, a composition, a mixture, a process, a method, an article,or an apparatus that comprises a list of elements is not necessarilylimited to only those elements but can include other elements notexpressly listed or inherent to such composition, mixture, process,method, article, or apparatus. Further, unless expressly stated to thecontrary, “or” refers to an inclusive or and not to an exclusive or. Forexample, a condition A or B is satisfied by any one of the following: Ais true (or present) and B is false (or not present), A is false (or notpresent) and B is true (or present), and both A and B are true (orpresent).

As used herein, the conjunctive term “and/or” between multiple recitedelements is understood as encompassing both individual and combinedoptions. For instance, where two elements are conjoined by “and/or,” afirst option refers to the applicability of the first element withoutthe second. A second option refers to the applicability of the secondelement without the first. A third option refers to the applicability ofthe first and second elements together. Any one of these options isunderstood to fall within the meaning, and therefore satisfy therequirement of the term “and/or” as used herein. Concurrentapplicability of more than one of the options is also understood to fallwithin the meaning, and therefore satisfy the requirement of the term“and/or.”

As used herein, the term “consists of,” or variations such as “consistof” or “consisting of,” as used throughout the specification and claims,indicate the inclusion of any recited integer or group of integers, butthat no additional integer or group of integers can be added to thespecified method, structure, or composition.

As used herein, the term “consists essentially of,” or variations suchas “consist essentially of” or “consisting essentially of,” as usedthroughout the specification and claims, indicate the inclusion of anyrecited integer or group of integers, and the optional inclusion of anyrecited integer or group of integers that do not materially change thebasic or novel properties of the specified method, structure orcomposition. See M.P.E.P. § 2111.03.

As used herein, “subject” or “patient” means any animal, preferably amammal, most preferably a human, to whom will be or has beenadministered a vaccine by a method according to an embodiment of theinvention. The term “mammal” as used herein, encompasses any mammal.Examples of mammals include, but are not limited to, cows, horses,sheep, pigs, cats, dogs, mice, rats, rabbits, guinea pigs, monkeys,humans, etc., more preferably a human.

As used herein, “a method of providing safe administration” means amethod of administration that is effective in generating an immuneresponse against a Zika virus without causing unacceptable adverseevents, when administered to a subject.

As used herein, the phrases “unacceptable adverse events” and“unacceptable adverse reaction” shall mean all harm or undesiredoutcomes associated with or caused by administration of a pharmaceuticalcomposition or therapeutic, and the harm or undesired outcome reachessuch a level of severity that a regulatory agency deems thepharmaceutical composition or therapeutic unacceptable for the proposeduse. Examples of unacceptable adverse events or reactions when used inthe context of administration of adenoviral particles comprising anucleic acid molecule encoding a Zika virus antigen can include, but isnot limited to, swelling, injection side pain, headache, malaise, muscleache, nausea, and fever.

In certain embodiments, “safe treatment” and “safe administration” whenused with respect to administration of adenoviral vectors comprising anucleic acid molecule encoding a Zika virus antigen means reducedadverse events including, but not limited to, swelling, injection sidepain, headache, malaise, muscle ache, nausea, and fever.

The terms “identical” or percent “identity,” in the context of two ormore nucleic acids or polypeptide sequences (e.g., Zika antigenpolypeptides and polynucleotides that encode them), refer to two or moresequences or subsequences that are the same or have a specifiedpercentage of amino acid residues or nucleotides that are the same, whencompared and aligned for maximum correspondence, as measured using oneof the following sequence comparison algorithms or by visual inspection.

For sequence comparison, typically one sequence acts as a referencesequence, to which test sequences are compared. When using a sequencecomparison algorithm, test and reference sequences are input into acomputer, subsequence coordinates are designated, if necessary, andsequence algorithm program parameters are designated. The sequencecomparison algorithm then calculates the percent sequence identity forthe test sequence(s) relative to the reference sequence, based on thedesignated program parameters.

Optimal alignment of sequences for comparison can be conducted, e.g., bythe local homology algorithm of Smith & Waterman, Adv. Appl. Math. 2:482(1981), by the homology alignment algorithm of Needleman & Wunsch, J.Mol. Biol. 48:443 (1970), by the search for similarity method of Pearson& Lipman, Proc. Nat'l. Acad. Sci. USA 85:2444 (1988), by computerizedimplementations of these algorithms (GAP, BESTFIT, FASTA, and TFASTA inthe Wisconsin Genetics Software Package, Genetics Computer Group, 575Science Dr., Madison, Wis.), or by visual inspection (see generally,Current Protocols in Molecular Biology, F. M. Ausubel et al., eds.,Current Protocols, a joint venture between Greene Publishing Associates,Inc. and John Wiley & Sons, Inc., (1995 Supplement) (Ausubel)).

Examples of algorithms that are suitable for determining percentsequence identity and sequence similarity are the BLAST and BLAST 2.0algorithms, which are described in Altschul et al. (1990) J. Mol. Biol.215: 403-410 and Altschul et al. (1997) Nucleic Acids Res. 25:3389-3402, respectively. Software for performing BLAST analyses ispublicly available through the National Center for BiotechnologyInformation. This algorithm involves first identifying high scoringsequence pairs (HSPs) by identifying short words of length W in thequery sequence, which either match or satisfy some positive-valuedthreshold score T when aligned with a word of the same length in adatabase sequence. T is referred to as the neighborhood word scorethreshold (Altschul et al, supra). These initial neighborhood word hitsact as seeds for initiating searches to find longer HSPs containingthem. The word hits are then extended in both directions along eachsequence for as far as the cumulative alignment score can be increased.

Cumulative scores are calculated using, for nucleotide sequences, theparameters M (reward score for a pair of matching residues; always >0)and N (penalty score for mismatching residues; always <0). For aminoacid sequences, a scoring matrix is used to calculate the cumulativescore. Extension of the word hits in each direction are halted when: thecumulative alignment score falls off by the quantity X from its maximumachieved value; the cumulative score goes to zero or below, due to theaccumulation of one or more negative-scoring residue alignments; or theend of either sequence is reached. The BLAST algorithm parameters W, T,and X determine the sensitivity and speed of the alignment. The BLASTNprogram (for nucleotide sequences) uses as defaults a wordlength (W) of11, an expectation (E) of 10, M=5, N=−4, and a comparison of bothstrands. For amino acid sequences, the BLASTP program uses as defaults awordlength (W) of 3, an expectation (E) of 10, and the BLOSUM62 scoringmatrix (see Henikoff & Henikoff, Proc. Natl. Acad. Sci. USA 89:10915(1989)).

In addition to calculating percent sequence identity, the BLASTalgorithm also performs a statistical analysis of the similarity betweentwo sequences (see, e.g., Karlin & Altschul, Proc. Nat'l. Acad. Sci. USA90:5873-5787 (1993)). One measure of similarity provided by the BLASTalgorithm is the smallest sum probability (P(N)), which provides anindication of the probability by which a match between two nucleotide oramino acid sequences would occur by chance. For example, a nucleic acidis considered similar to a reference sequence if the smallest sumprobability in a comparison of the test nucleic acid to the referencenucleic acid is less than about 0.1, more preferably less than about0.01, and most preferably less than about 0.001.

A further indication that two nucleic acid sequences or polypeptides aresubstantially identical is that the polypeptide encoded by the firstnucleic acid is immunologically cross reactive with the polypeptideencoded by the second nucleic acid, as described below. Thus, apolypeptide is typically substantially identical to a secondpolypeptide, for example, where the two peptides differ only byconservative substitutions. Another indication that two nucleic acidsequences are substantially identical is that the two moleculeshybridize to each other under stringent conditions.

As used herein, the term “immune response” or “protective immuneresponse” means that the vaccinated subject is able to control aninfection (e.g., a Zika virus (ZIKV) infection) with the pathogenicagent against which the vaccination was done (e.g., a ZIKV antigen). Thepathogenic agent can, for example, be an antigenic gene product orantigenic protein, or a fragment thereof. Usually, the subject havingdeveloped an “immune response” or a “protective immune response”develops only mild to moderate clinical symptoms or no symptoms at all.Usually, a subject in which an “immune response” or “protective immuneresponse” against a Zika virus has been generated, will not developdisease manifestations or those disease manifestations will be milder,and ultimately the subject will not die as a result of the infectionwith said virus. In addition, a subject in which an “immune response” or“protective immune response” against a Zika virus has been generated,will have a reduced chance of brain abnormalities in her infants exposedto the virus in utero.

By “generating an immune response” or “promoting an immune response” or“provoking an immune response” is meant eliciting a humoral response(e.g., the production of antibodies) or a cellular response (e.g., theactivation of T cells, macrophages, neutrophils, and/or natural killercells) directed against, for example, one or more infective agents(e.g., a virus (e.g., a ZIKV)) or protein targets in a subject to whichthe pharmaceutical composition (e.g., an immunogenic composition orvaccine) has been administered.

By “immunogen” or “antigen” is meant any polypeptide that can induce animmune response in a subject upon administration. In some embodiments,the immunogen or antigen is encoded by a nucleic acid molecule that maybe incorporated into, for example, a polynucleotide or vector of theinvention, for subsequent expression of the immunogen or antigen (e.g.,a gene product of interest, or fragment thereof (e.g., a polypeptide)).In some embodiments, the immunogen is derived from a ZIKV (e.g., a ZIKVfrom the Asian and/or African lineage (e.g., ZIKV strain BeH815744(accession number KU365780 (SEQ ID NOs: 15-16))). In some embodiments,the immunogen is administered in the context of a nucleic acid moleculeexpressing a polypeptide that is derived from a ZIKV (e.g., a ZIKV fromthe Asian and/or African lineage (e.g., ZIKV strain BeH815744 (accessionnumber KU365780 (SEQ ID NOs: 15-16))).

The term “immunogenic composition” as used herein, is defined asmaterial used to generate an immune response and may confer immunityafter administration of the immunogenic composition to a subject.

By “isolated” is meant separated, recovered, or purified from acomponent of its natural environment. For example, a nucleic acidmolecule or polypeptide of the invention may be isolated from acomponent of its natural environment by 1% (2%, 3%, 4%, 5%, 6%, 7%, 8%9% 10%, 20%, 30%, 40%, 50%, 60% 70%, 80%, or 90%) or more.

Nucleic Acid Molecules, Polypeptides, and Vectors of the Invention

In PCT/US2017/36900, filed on Jun. 9, 2017, entitled “Compositions andMethods for Preventing and Treating Zika Virus Infection,” which isincorporated by reference in its entirety, disclosed are Zika virus(ZIKV) polypeptides that can be used to elicit protective immuneresponses against a ZIKV infection when administered to a subject (e.g.,a mouse or monkey) infected with or likely to be exposed to a ZIKVinfection. The ZIKV polypeptides for use in pharmaceutical compositionsprepared for administration can include a M-Env, prM-Env, prM-Env.dTM,prM-Env.dStem, Env, Env.dTM, and/or Env.dStem or a portion thereof.Alternatively, the ZIKV polypeptides can be encoded for by a nucleicacid molecule comprised within a vector (e.g., an adenoviral vector).

Table 1 provides the ZIKV derived polypeptide and polynucleotidemolecules that can be used in pharmaceutical compositions of theinvention for the safe administration and prevention of a Zika virusinfection.

TABLE 1 ZIKV derived polynucleotide and polypeptide molecules SEQ ID NO.Region of ZIKV polynucleotide polypeptide M-Env 1 2 prM-Env.dTM 3 4prM-Env.dStem 5 6 Env 7 8 Env.dTM 9 10 Env.dStem 11 12 prM-Env (fulllength) 24 25 prM-Env with JEV Stem/TM 26 27

The nucleic acid molecules of the invention (Table 1) were designedbased on the Zika virus (ZIKV) strain BeH815744 (accession numberKU365780 (SEQ ID NOs: 15-16)). The nucleic acid molecules of theinvention encode regions of the Zika virus (ZIKV) polyprotein, forexample, the membrane and envelope (M-Env) proteins, the pre-membraneand envelope (prM-Env) region, the Env protein alone, or deletionmutants of the prM-Env or Env regions in which the transmembrane (TM) orStem region have been removed. The nucleic acid molecules of theinvention M-Env (SEQ ID NO: 1), prM-Env.dTM (SEQ ID NO: 3),prM-Env.dStem (SEQ ID NO: 5), Env (SEQ ID NO: 7), Env.dTM (SEQ ID NO:9), and Env.dStem (SEQ ID NO: 11) have been optimized relative to thewild-type BeH815744 nucleotide sequences for improved expression in hostcells (e.g., mammalian (e.g., human) host cells) and particle formation,and encode the polypeptides set forth in SEQ ID NOs: 2, 4, 6, 8, 10, or12, respectively (Table 1). Optimization can include the addition of aleader sequence, such as a Japanese encephalitis virus (JEV) leadersequence (e.g., SEQ ID NO: 13), restriction site (e.g., SEQ ID NOs:21-22), and/or a Kozak sequence (e.g., SEQ ID NO: 23).

The prM-Env (full length) (e.g., SEQ ID NOs: 24-25) contains thefull-length sequence of the prM-Env region, while prM-Env with JEVStem/TM (e.g., SEQ ID NOs: 26-27) includes the ZIKV prM signal region ofJapanese encephalitis virus (JEV) with the final 98 amino acidscomprising the stem and transmembrane regions exchanged withcorresponding JEV sequences.

The nucleic acid molecules have a nucleotide sequence with at least 85%(e.g., at least 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%,97%, 98%, 99% or 100%) sequence identity to, all or a portion of any oneof SEQ ID NOs: 1, 3, 5, 7, 9, or 11, or a complementary sequencethereof. Alternatively, an isolated nucleic acid molecule has anucleotide sequence that encodes a ZIKV polypeptide with at least 85%(e.g., at least 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%,97%, 98%, 99% or 100%) sequence identity to an amino acid sequence ofany one of SEQ ID NOs: 2, 4, 6, 8, 10, or 12.

The nucleic acid molecules of the invention may be further optimized,such as by codon optimization, for expression in a targeted mammaliansubject (e.g., human).

The nucleic acid molecules can also be inserted into expression vectors,such as an adenovirus vector and incorporated into compositions of theinvention. The terms “adenovirus vector” and “adenoviral vector” and“adenoviral particles” are used interchangeably and refer to agenetically-engineered adenovirus that is designed to insert apolynucleotide of interest (e.g., a polynucleotide encoding a ZIKVantigen of the invention) into a eukaryotic cell, such that thepolynucleotide is subsequently expressed. Examples of adenoviruses thatcan be used as a viral vector of the invention include those having, orderived from, the serotypes Ad2, Ad5, Ad11, Ad12, Ad24, Ad26, Ad34,Ad35, Ad40, Ad48, Ad49, Ad50, Ad52 (e.g., RhAd52), and Pan9 (also knownas AdC68); these vectors can be derived from, for example, human,chimpanzee (e.g., ChAdl, ChAd3, ChAd7, ChAd8, ChAd21, ChAd22, ChAd23,ChAd24, ChAd25, ChAd26, ChAd27.1, ChAd28.1, ChAd29, ChAd30, ChAd31.1,ChAd32, ChAd33, ChAd34, ChAd35.1, ChAd36, ChAd37.2, ChAd39, ChAd40.1,ChAd41.1, ChAd42.1, ChAd43, ChAd44, ChAd45, ChAd46, ChAd48, ChAd49,ChAd49, ChAd50, ChAd67, or SA7P), or rhesus adenoviruses (e.g., rhAd51,rhAd52, or rhAd53).

“Nucleic acid molecule” or “polynucleotide,” as used interchangeablyherein, refer to polymers of nucleotides of any length, and include DNAand RNA. The nucleotides can be deoxyribonucleotides, ribonucleotides,modified nucleotides or bases, and/or their analogs, or any substratethat can be incorporated into a polymer by DNA or RNA polymerase, or bya synthetic reaction. A polynucleotide can comprise modifiednucleotides, such as methylated nucleotides and their analogs. Ifpresent, modification to the nucleotide structure can be imparted beforeor after assembly of the polymer. The sequence of nucleotides can beinterrupted by non-nucleotide components. A polynucleotide can befurther modified after synthesis, such as by conjugation with a label.

By “heterologous nucleic acid molecule” is meant a nucleotide sequencethat can encode proteins derived or obtained from pathogenic organisms,such as viruses, which can be incorporated into a polynucleotide orvector of the invention. Heterologous nucleic acids can also encodesynthetic or artificial proteins, such as immunogenic epitopes,constructed to induce immunity. An example of a heterologous nucleicacid molecule is one that encodes one or more immunogenic peptides orpolypeptides derived from a Zika virus (ZIKV). The heterologous nucleicacid molecule is one that is not normally associated with the othernucleic acid molecules found in the polynucleotide or vector into whichthe heterologous nucleic acid molecule is incorporated.

A “nucleic acid vaccine” or “DNA vaccine” refers to a vaccine thatincludes a heterologous nucleic acid molecule under the control of apromoter for expression in a subject. The heterologous nucleic acidmolecule can be incorporated into an expression vector, such as aplasmid or an adenoviral vector.

The term “vaccine” as used herein, is defined as material used toprovoke an immune response and that confers immunity for a period oftime after administration of the vaccine to a subject.

A “promoter” is a nucleic acid sequence enabling the initiation of thetranscription of a gene sequence in a messenger RNA, such transcriptionbeing initiated with the binding of an RNA polymerase on or nearby thepromoter.

A nucleic acid is “operably linked” when it is placed into a structuralor functional relationship with another nucleic acid sequence. Forexample, one segment of DNA can be operably linked to another segment ofDNA if they are positioned relative to one another on the samecontiguous DNA molecule and have a structural or functionalrelationship, such as a promoter or enhancer that is positioned relativeto a coding sequence so as to facilitate transcription of the codingsequence; a ribosome binding site that is positioned relative to acoding sequence so as to facilitate translation; or a pre-sequence orsecretory leader that is positioned relative to a coding sequence so asto facilitate expression of a pre-protein (e.g., a pre-protein thatparticipates in the secretion of the encoded polypeptide). In otherexamples, the operably linked nucleic acid sequences are not contiguous,but are positioned in such a way that they have a functionalrelationship with each other as nucleic acids or as proteins that areexpressed by them. Enhancers, for example, do not have to be contiguous.Linking can be accomplished by ligation at convenient restriction sitesor by using synthetic oligonucleotide adaptors or linkers.

The polypeptides encoded by vectors useful for the invention are ZIKVpolypeptides corresponding to, for example, the membrane and envelope(M-Env) proteins, the pre-membrane and envelope (prM-Env) region, theEnv protein alone, or deletion mutants of the prM-Env or

Env regions in which the transmembrane (TM) or Stem region has beenremoved. Exemplary polypeptides encoded by vectors useful for theinvention include M-Env (SEQ ID NO: 2), prM-Env.dTM (SEQ ID NO: 4),prM-Env.dStem (SEQ ID NO: 6), Env (SEQ ID NO: 8), Env.dTM (SEQ ID NO:10), and Env.dStem (SEQ ID NO: 12) and variants having at least 85%(e.g., at least 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%,97%, 98%, 99% or 100%) sequence identity to, all or a portion of any oneof SEQ ID NOs: 2, 4, 6, 8, 10, or 12. The polypeptides of the inventionmay include at least 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90,100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 1100, 1200, 1300,1400, 1500, 1600, 1700, or 1800 or more continuous or non-continuousamino acids of any one of SEQ ID NOs: 2, 4, 6, 8, 10, or 12.

Polypeptides encoded by vectors useful for the invention can alsoinclude a signal/leader sequence, such as a Japanese encephalitis virus(JEV) signal/leader sequence (SEQ ID NO: 14). The polypeptides can alsobe isolated from other components (e.g., components with which thepolypeptides are natively associated) and incorporated into compositionsof the invention.

By “portion” or “fragment” is meant a part of a whole. A portion cancomprise at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 95% ofthe entire length of a polynucleotide or polypeptide sequence region.For polynucleotides, for example, a portion can include at least 5, 6,7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 200, 300, 400, 500,600, 700, 800, 900, 1000, 1100, 1200, 1300, 1400, 1500, 1600, 1700, 1800or more contiguous nucleotides of a reference polynucleotide molecule.For polypeptides, for example, a portion can include at least 5, 6, 7,8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 50, 75, 90, 100,125, 150, 175, 200, 225, 250, 275, 300, 325, 350, 375, 400, 425, 450,475, 500, 525, 550, 575, or 600 or more continuous amino acids of areference polypeptide molecule.

In some instances, a fragment of a nucleic acid molecule can include atleast 20, 30, 40, 50, 60, 70, 80, 90, 100, 200, 300, 400, 500, 600, 700,800, 900, 1000, 1100, 1200, 1300, 1400, 1500, 1600, 1700 or moreconsecutive nucleotides of the polynucleotide M-Env (SEQ ID NO: 1). Insome instances, a fragment of a nucleic acid molecule can include atleast 20, 30, 40, 50, 60, 70, 80, 90, 100, 200, 300, 400, 500, 600, 700,800, 900, 1000, 1100, 1200, 1300, 1400, 1500, or more consecutivenucleotides of the polynucleotide prM-Env.dTM (SEQ ID NO: 3). In someinstances, a fragment of a nucleic acid molecule can include at least20, 30, 40, 50, 60, 70, 80, 90, 100, 200, 300, 400, 500, 600, 700, 800,900, 1000, 1100, 1200, 1300, 1400, or more consecutive nucleotides ofthe polynucleotide prM-Env.dStem (SEQ ID NO: 5). In some instances, afragment of a nucleic acid molecule can include at least 20, 30, 40, 50,60, 70, 80, 90, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 1100,1200, 1300, 1400, 1500, or more consecutive nucleotides of thepolynucleotide Env (SEQ ID NO: 7). In some instances, a fragment of anucleic acid molecule can include at least 20, 30, 40, 50, 60, 70, 80,90, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 1100, 1200, 1300,or more consecutive nucleotides of the polynucleotide Env.dTM (SEQ IDNO: 9). In some instances, a fragment of a nucleic acid molecule caninclude at least 20, 30, 40, 50, 60, 70, 80, 90, 100, 200, 300, 400,500, 600, 700, 800, 900, 1000, 1100, 1200, or more consecutivenucleotides of the polynucleotide Env.dStem (SEQ ID NO: 11). In someinstances, a fragment of a nucleic acid molecule can include at least 5,6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 200, 300, 400, 500,600, 700, 800, 900, 1000, 1100, 1200, 1300, 1400, 1500, 1600, 1700,1800, 1900, 2000, or more consecutive nucleotides of the polynucleotideprM-Env (full length) (SEQ ID NO: 24). In some instances, a fragment ofa nucleic acid molecule can include at least 20, 30, 40, 50, 60, 70, 80,90, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 1100, 1200, 1300,1400, 1500, 1600, 1700, 1800, 1900, 2000, or more consecutivenucleotides of the polynucleotide prM-Env with JEV Stem/TM (SEQ ID NO:26).

In some instances, a fragment of a polypeptide can include at least 20,25, 50, 75, 90, 100, 125, 150, 175, 200, 225, 250, 275, 300, 325, 350,375, 400, 425, 450, 475, 500, 525, 550, or more consecutive amino acidsof polypeptide M-Env (SEQ ID NO: 2). In some instances, a fragment of apolypeptide can include at least 20, 25, 50, 75, 90, 100, 125, 150, 175,200, 225, 250, 275, 300, 325, 350, 375, 400, 425, 450, 475, 500, or moreconsecutive amino acids of polypeptide prM-Env.dTM (SEQ ID NO: 4). Insome instances, a fragment of a polypeptide can include at least 20, 25,50, 75, 90, 100, 125, 150, 175, 200, 225, 250, 275, 300, 325, 350, 375,400, 425, 450, 475, or more consecutive amino acids of polypeptideprM-Env.dStem (SEQ ID NO: 6). In some instances, a fragment of apolypeptide can include at least 20, 25, 50, 75, 90, 100, 125, 150, 175,200, 225, 250, 275, 300, 325, 350, 375, 400, 425, 450, 475, 500, or moreconsecutive amino acids of polypeptide Env (SEQ ID NO: 8). In someinstances, a fragment of a polypeptide can include at least 20, 25, 50,75, 90, 100, 125, 150, 175, 200, 225, 250, 275, 300, 325, 350, 375, 400,425, or more consecutive amino acids of polypeptide Env.dTM (SEQ ID NO:10). In some instances, a fragment of a polypeptide can include at least20, 25, 50, 75, 90, 100, 125, 150, 175, 200, 225, 250, 275, 300, 325,350, 375, 400, or more consecutive amino acids of polypeptide Env.dStem(SEQ ID NO: 12). In some instances, a fragment of a polypeptide caninclude at least 20, 25, 50, 75, 90, 100, 125, 150, 175, 200, 225, 250,275, 300, 325, 350, 375, 400, 425, 450, 475, 500, 525, 550, 575, 600,625, 650, 675, or more consecutive amino acids of polypeptide prM-Env(full length) (SEQ ID NO: 25). In some instances, a fragment of apolypeptide can include at least 20, 25, 50, 75, 90, 100, 125, 150, 175,200, 225, 250, 275, 300, 325, 350, 375, 400, 425, 450, 475, 500, 525,550, 575, 600, 625, 650, 675, or more consecutive amino acids ofpolypeptide prM-Env with JEV Stem/TM (SEQ ID NO: 27).

In some instances, administration of a fragment of a polynucleotide(e.g., SEQ ID NOs: 1, 3, 5, 7, 9, 11, 24, and/or 26) and/or apolypeptide (e.g., SEQ ID NOs: 2, 4, 6, 8, 10, 12, 25, and/or 27) of theinvention to a subject can illicit an immune response in the subject.

The invention also features recombinant vectors including any one ormore of the polynucleotides described above. The vectors of theinvention can be used to deliver a nucleic acid expressing an immunogenof the invention (e.g., one of more of SEQ ID NOs: 2, 4, 6, 8, 10, or 12or variants thereof, having at least 85-99% sequence identity thereto,for example at least greater than 90% sequence identity thereto), andinclude mammalian, viral, and bacterial expression vectors. Themammalian, viral, and bacterial vectors of the invention can begenetically modified to contain one or more nucleic acid sequences setforth in SEQ ID NOs: 1, 3, 5, 7, 9, or 11 or variants thereof, having atleast 85-99% sequence identity thereto, for example at least greaterthan 90% sequence identity thereto, and complements thereof.

Promoters and other expression regulation signals can be selected to becompatible with the host cell for which expression is designed. Forexample, mammalian promoters include the metallothionein promoter, whichcan be induced in response to heavy metals, such as cadmium, and theβ-actin promoter. A viral promoter, which can be obtained from thegenome of a virus, such as, for example, polyoma virus, fowlpox virus,adenovirus (A), bovine papilloma virus, avian sarcoma virus,cytomegalovirus (CMV), a retrovirus, hepatitis-B virus, and Simian Virus40 (SV40), and human papillomavirus (HPV), can also be used. Thesepromoters are well known and readily available in the art.

A preferred promoter element is the CMV immediate early promoter. Insome embodiments, the expression plasmid is pcDNA3.1+(Invitrogen, CA,USA). In some embodiments, the expression vector is a viral vector, suchas a vector derived from adenovirus or poxvirus.

Viral genomes provide a rich source of vectors that can be used for theefficient delivery of exogenous genes into the genome of a cell (e.g., aeukaryotic or prokaryotic cell). Viral genomes are particularly usefulvectors for gene delivery because the polynucleotides contained withinsuch genomes are typically incorporated into the genome of a target cellby generalized or specialized transduction. These processes occur aspart of the natural viral replication cycle, and do not require addedproteins or reagents in order to induce gene integration. Examples ofviral vectors that can be used to deliver a nucleic acid expressing animmunogen of the invention (e.g., one of more of SEQ ID NOs: 2, 4, 6, 8,10, or 12 or variants thereof having at least 85-99% sequence identitythereto, for example at least greater than 90% sequence identitythereto) include adenovirus (e.g., Ad2, Ad5, Ad11, Ad12, Ad24, Ad26,Ad34, Ad35, Ad40, Ad48, Ad49, Ad50, Ad52 (e.g., RhAd52), and Pan9 (alsoknown as AdC68)). These adenovirus vectors can be derived from, forexample, human, chimpanzee (e.g., ChAdl, ChAd3, ChAd7, ChAd8, ChAd21,ChAd22, ChAd23, ChAd24, ChAd25, ChAd26, ChAd27.1, ChAd28.1, ChAd29,ChAd30, ChAd31.1, ChAd32, ChAd33, ChAd34, ChAd35.1, ChAd36, ChAd37.2,ChAd39, ChAd40.1, ChAd41.1, ChAd42.1, ChAd43, ChAd44, ChAd45, ChAd46,ChAd48, ChAd49, ChAd49, ChAd50, ChAd67, or SA7P), or rhesusadenoviruses. The viral vector can be used to infect cells of a subject,which, in turn, promotes the translation of the heterologous gene(s) ofthe viral vector into the immunogens of the invention. For example, aviral vector of the invention can be genetically modified to contain oneor more nucleic acid sequences set forth in SEQ ID NOs: 1, 3, 5, 7, 9,or 11 or variants thereof having at least 85-99% sequence identitythereto, for example at least greater than 90% sequence identitythereto, and complements thereof.

Adenoviral vectors disclosed in International Patent ApplicationPublications WO2006/040330 and WO2007/104792, each incorporated byreference herein, are particularly useful as vectors of the invention.These adenoviral vectors can encode and/or deliver one or more of theimmunogens of the invention (e.g., ZIKV polypeptides) to treat a subjecthaving a pathological condition associated with a viral infection (e.g.,a ZIKV infection). In some embodiments, one or more recombinantadenovirus vectors can be administered to the subject in order toexpress more than one type of immunogen (e.g., ZIKV polypeptide) of theinvention.

Adenoviruses

An adenovirus according to the invention belongs to the family of theAdenoviridae and preferably is one that belongs to the genusMastadenovirus. It can be a human adenovirus, but also an adenovirusthat infects other species, including but not limited to a bovineadenovirus (e.g. bovine adenovirus 3, BAdV3), a canine adenovirus (e.g.CAdV2), a porcine adenovirus (e.g. PAdV3 or 5), or a simian adenovirus(which includes a monkey adenovirus and an ape adenovirus, such as achimpanzee adenovirus or a gorilla adenovirus). Preferably, theadenovirus is a human adenovirus (HAdV, or AdHu; in the invention ahuman adenovirus is meant if referred to Ad without indication ofspecies, e.g. the brief notation “Ad5” means the same as HAdV5, which ishuman adenovirus serotype 5), or a simian adenovirus such as chimpanzeeor gorilla adenovirus (ChAd, AdCh, or SAdV).

Most advanced studies have been performed using human adenoviruses, andhuman adenoviruses are preferred according to certain aspects of theinvention. In certain preferred embodiments, the recombinant adenovirusaccording to the invention is based upon a human adenovirus. Inpreferred embodiments, the recombinant adenovirus is based upon a humanadenovirus serotype 5, 11, 26, 34, 35, 48, 49 or 50. According to aparticularly preferred embodiment of the invention, an adenovirus is ahuman adenovirus of one of the serotypes 26 or 35.

An advantage of these serotypes is a low seroprevalence and/or lowpre-existing neutralizing antibody titers in the human population.Preparation of rAd26 vectors is described, for example, in WO2007/104792 and in Abbink et al., (2007) Virol 81(9):4654-63, both ofwhich are incorporated by reference herein in their entirety. Exemplarygenome sequences of Ad26 are found in GenBank Accession EF 153474 and inSEQ ID NO:1 of WO 2007/104792. Preparation of rAd35 vectors isdescribed, for example, in U.S. Pat. No. 7,270,811, in WO 00/70071, andin Vogels et al., (2003) J Virol 77(15): 8263-71, all of which areincorporated by reference herein in their entirety. Exemplary genomesequences of Ad35 are found in GenBank Accession AC_000019 and in FIG. 6of WO 00/70071.

Simian adenoviruses generally also have a low seroprevalence and/or lowpre-existing neutralizing antibody titers in the human population, and asignificant amount of work has been reported using chimpanzee adenovirusvectors (e.g. U.S. Pat. No. 6,083,716; WO 2005/071093; WO 2010/086189;WO 2010085984; Farina et al, 2001, J Virol 75: 11603-13; Cohen et al,2002, J Gen Virol 83: 151-55; Kobinger et al, 2006, Virology 346:394-401; Tatsis et al., 2007, Molecular Therapy 15: 608-17; see alsoreview by Bangari and Mittal, 2006, Vaccine 24: 849-62; and review byLasaro and Ertl, 2009, Mol Ther 17: 1333-39). Hence, in other preferredembodiments, the recombinant adenovirus according to the invention isbased upon a simian adenovirus, e.g. a chimpanzee adenovirus. In certainembodiments, the recombinant adenovirus is based upon simian adenovirustype 1, 7, 8, 21, 22, 23, 24, 25, 26, 27.1, 28.1, 29, 30, 31.1, 32, 33,34, 35.1, 36, 37.2, 39, 40.1, 41.1, 42.1, 43, 44, 45, 46, 48, 49, 50 orSA7P.

Adenoviral Vector rAd26

In a preferred embodiment according to the invention the adenoviralvectors comprise capsid proteins from two rare serotypes: Ad26 or Ad35.In the typical embodiment, the vector is an rAd26 virus.

Thus, the vectors that can be used in the invention comprise an Ad26capsid protein (e.g., a fiber, penton or hexon protein). One of skillwill recognize that it is not necessary that an entire Ad26 capsidprotein be used in the vectors of the invention. Thus, chimeric capsidproteins that include at least a part of an Ad26 capsid protein can beused in the vectors of the invention. The vectors of the invention canalso comprise capsid proteins in which the fiber, penton, and hexonproteins are each derived from a different serotype, so long as at leastone capsid protein is derived from Ad26. In preferred embodiments, thefiber, penton and hexon proteins are each derived from Ad26.

One of skill will recognize that elements derived from multipleserotypes can be combined in a single recombinant adenovirus vector.Thus, a chimeric adenovirus that combines desirable properties fromdifferent serotypes can be produced. Thus, in some embodiments, achimeric adenovirus of the invention can combine the absence ofpre-existing immunity of the Ad26 serotypes with characteristics such astemperature stability, assembly, anchoring, production yield, redirectedor improved infection, stability of the DNA in the target cell, and thelike.

In certain embodiments the recombinant adenovirus vector useful in theinvention is derived mainly or entirely from Ad26 (i.e., the vector isrAd26). In some embodiments, the adenovirus is replication deficient,e.g. because it contains a deletion in the E1 region of the genome. Forthe adenoviruses of the invention, being derived from Ad26, it istypical to exchange the E4-orf6 coding sequence of the adenovirus withthe E4-orf6 of an adenovirus of human subgroup C such as Ad5. Thisallows propagation of such adenoviruses in well-known complementing celllines that express the E1 genes of Ad5, such as for example 293 cells,PER.C6 cells, and the like (see, e.g. Havenga et al, 2006, J Gen Virol87: 2135-43; WO 03/104467). In certain embodiments, the adenovirus is ahuman adenovirus of serotype 35, with a deletion in the E1 region intowhich the nucleic acid encoding the antigen has been cloned, and with anE4 orf6 region of Ad5. In certain embodiments, the adenovirus is a humanadenovirus of serotype 26, with a deletion in the E1 region into whichthe nucleic acid encoding the antigen has been cloned, and with an E4orf6 region of Ad5. For the Ad35 adenovirus, it is typical to retain the3′ end of the E1B 55K open reading frame in the adenovirus, for instancethe 166 bp directly upstream of the pIX open reading frame or a fragmentcomprising this such as a 243 bp fragment directly upstream of the pIXstart codon, marked at the 5′ end by a Bsu36I restriction site, sincethis increases the stability of the adenovirus because the promoter ofthe pIX gene is partly residing in this area (see, e.g. Havenga et al,2006, supra; WO 2004/001032). The preparation of recombinant adenoviralvectors is well known in the art.

Preparation of rAd26 vectors is described, for example, in WO2007/104792 and in Abbink et al., (2007) Virol 81(9): 4654-63. Exemplarygenome sequences of Ad26 are found in GenBank Accession EF 153474 and inSEQ ID NO:1 of WO 2007/104792. Preparation of rAd35 vectors isdescribed, for example, in U.S. Pat. No. 7,270,811 and in Vogels et al.,(2003) J Virol 77(15): 8263-71. An exemplary genome sequence of Ad35 isfound in GenBank Accession AC_000019.

In an embodiment of the invention, the vectors useful for the inventioninclude those described in WO2012/082918, the disclosure of which isincorporated herein by reference in its entirety.

Typically, a vector useful in the invention is produced using a nucleicacid comprising the entire recombinant adenoviral genome (e.g., aplasmid, cosmid, or baculovirus vector). Thus, the invention alsoprovides isolated nucleic acid molecules that encode the adenoviralvectors of the invention. The nucleic acid molecules of the inventioncan be in the form of RNA or in the form of DNA obtained by cloning orproduced synthetically. The DNA can be double-stranded orsingle-stranded.

The adenovirus vectors useful in the invention are typically replicationdefective. In these embodiments, the virus is renderedreplication-defective by deletion or inactivation of regions critical toreplication of the virus, such as the E1 region. The regions can besubstantially deleted or inactivated by, for example, inserting the geneof interest (usually linked to a promoter). In some embodiments, thevectors of the invention can contain deletions in other regions, such asthe E2, E3 or E4 regions or insertions of heterologous genes linked to apromoter. For E2- and/or E4-mutated adenoviruses, generally E2- and/orE4-complementing cell lines are used to generate recombinantadenoviruses. Mutations in the E3 region of the adenovirus need not becomplemented by the cell line, since E3 is not required for replication.

A packaging cell line is typically used to produce sufficient amount ofadenovirus vectors of the invention. A packaging cell is a cell thatcomprises those genes that have been deleted or inactivated in areplication-defective vector, thus allowing the virus to replicate inthe cell. Suitable cell lines include, for example, PER.C6, 911, 293,and E1 A549.

Pharmaceutical Compositions

In another general aspect, the invention relates to pharmaceuticalcompositions comprising adenoviral vectors (or adenoviral particles)comprising a nucleic acid molecule encoding a Zika virus antigen of theinvention and a pharmaceutically acceptable carrier. Adenoviral vectors(or particles) of the invention and compositions comprising them arealso useful in the manufacture of a medicament for therapeuticapplications mentioned herein.

By “pharmaceutical composition” is meant any composition that contains atherapeutically or biologically active agent, such as an immunogeniccomposition or vaccine of the invention (e.g., an adenoviral particlecomprising a ZIKV nucleic acid molecule and/or polypeptide of theinvention), preferably including a nucleotide sequence encoding anantigenic gene product of interest, or fragment thereof, that issuitable for administration to a subject and that treats or prevents adisease (e.g., ZIKV infection) or reduces or ameliorates one or moresymptoms of the disease (e.g., ZIKV viral titer, viral spread,infection, and/or cell fusion)). For the purposes of this invention,pharmaceutical compositions include vaccines, and pharmaceuticalcompositions suitable for delivering a therapeutic or biologicallyactive agent can include, for example, tablets, gelcaps, capsules,pills, powders, granulates, suspensions, emulsions, solutions, gels,hydrogels, oral gels, pastes, eye drops, ointments, creams, plasters,drenches, delivery devices, suppositories, enemas, injectables,implants, sprays, or aerosols. Any of these formulations can be preparedby well-known and accepted methods of art. See, for example, Remington:The Science and Practice of Pharmacy (21^(st) ed.), ed. A. R. Gennaro,Lippincott Williams & Wilkins, 2005, and Encyclopedia of PharmaceuticalTechnology, ed. J. Swarbrick, Informa Healthcare, 2006, each of which ishereby incorporated by reference.

As used herein, the term “carrier” refers to any excipient, diluent,filler, salt, buffer, stabilizer, solubilizer, oil, lipid, lipidcontaining vesicle, microsphere, liposomal encapsulation, or othermaterial well known in the art for use in pharmaceutical formulations.It will be understood that the characteristics of the carrier, excipientor diluent will depend on the route of administration for a particularapplication. As used herein, the term “pharmaceutically acceptablecarrier” refers to a non-toxic material that does not interfere with theeffectiveness of a composition according to the invention or thebiological activity of a composition according to the invention.According to particular embodiments, in view of the present disclosure,any pharmaceutically acceptable carrier suitable for use in apharmaceutical composition can be used in the invention.

Pharmaceutically acceptable acidic/anionic salts for use in theinvention include, and are not limited to acetate, benzenesulfonate,benzoate, bicarbonate, bitartrate, bromide, calcium edetate, camsylate,carbonate, chloride, citrate, dihydrochloride, edetate, edisylate,estolate, esylate, fumarate, glyceptate, gluconate, glutamate,glycollylarsanilate, hexylresorcinate, hydrabamine, hydrobromide,hydrochloride, hydroxynaphthoate, iodide, isethionate, lactate,lactobionate, malate, maleate, mandelate, mesylate, methylbromide,methylnitrate, methylsulfate, mucate, napsylate, nitrate, pamoate,pantothenate, phosphate/diphosphate, polygalacturonate, salicylate,stearate, subacetate, succinate, sulfate, tannate, tartrate, teoclate,tosylate and triethiodide. Organic or inorganic acids also include, andare not limited to, hydriodic, perchloric, sulfuric, phosphoric,propionic, glycolic, methanesulfonic, hydroxyethanesulfonic, oxalic,2-naphthalenesulfonic, p-toluenesulfonic, cyclohexanesulfamic,saccharinic or trifluoroacetic acid.

Pharmaceutically acceptable basic/cationic salts include, and are notlimited to aluminum, 2-amino-2-hydroxymethyl-propane-1,3-diol (alsoknown as tris(hydroxymethyl)aminomethane, tromethane or “TRIS”),ammonia, benzathine, t-butylamine, calcium, chloroprocaine, choline,cyclohexylamine, diethanolamine, ethylenediamine, lithium, L-lysine,magnesium, meglumine, N-methyl-D-glucamine, piperidine, potassium,procaine, quinine, sodium, triethanolamine, or zinc.

In some embodiments of the invention, pharmaceutical compositions areprovided comprising the adenoviral vectors of the invention in an amountfrom about 1×10¹⁰, about 2×10¹⁰, about 3×10¹⁰, about 4×10¹⁰, about5×10¹⁰, about 6×10¹⁰, about 7×10¹⁰, about 8×10¹⁰, about 9×10¹⁰, about1×10¹¹, about 2×10¹¹, about 3×10¹¹, about 4×10¹¹, or about 5×10¹¹ viralvectors (or particles) per dose. In certain embodiments of theinvention, the pharmaceutical composition comprises about 1×10¹⁰adenoviral vectors (or particles) to about 5×10¹¹ adenoviral vectors (orparticles) per dose. In certain embodiments of the invention, thepharmaceutical composition comprises about 5×10¹⁰ adenoviral vectors (orparticles) to about 1×10¹¹ adenoviral vectors (or particles) per dose.In certain embodiments of the invention, the pharmaceutical compositioncomprises about 5×10¹⁰ adenoviral vectors (or particles) per dose. Incertain embodiments of the invention, the pharmaceutical compositioncomprises about 1×10¹¹ adenoviral vectors (or particles) per dose.

The pharmaceutical composition can have a pH from about 3.0 to about 10,for example from about 3 to about 7, or from about 5 to about 9. Theformulation can further comprise at least one ingredient selected fromthe group consisting of a buffer system, preservative(s), tonicityagent(s), chelating agent(s), stabilizer(s) and surfactant(s).

In certain embodiments, the subject is administered a single dose of thepharmaceutical composition. In certain embodiments, the subject isadministered a double dose of the pharmaceutical composition. Whenadministering a double dose, the first and second dose of thepharmaceutical composition can be administered to the subject about fourweeks, about eight weeks, about twelve weeks, about three months, aboutsix months, about nine months, about one year, or about two years apart.

The formulation of pharmaceutically active ingredients withpharmaceutically acceptable carriers is known in the art, e.g.,Remington: The Science and Practice of Pharmacy (e.g. 21st edition(2005), and any later editions). Non-limiting examples of additionalingredients include: buffers, diluents, solvents, tonicity regulatingagents, preservatives, stabilizers, and chelating agents. One or morepharmaceutically acceptable carriers can be used in formulating thepharmaceutical compositions of the invention.

In one embodiment of the invention, the pharmaceutical composition is aliquid formulation. A preferred example of a liquid formulation is anaqueous formulation, i.e., a formulation comprising water. The liquidformulation can comprise a solution, a suspension, an emulsion, amicroemulsion, a gel, and the like. An aqueous formulation typicallycomprises at least 50% w/w water, or at least 60%, 70%, 75%, 80%, 85%,90%, or at least 95% w/w of water.

In one embodiment, the pharmaceutical composition can be formulated asan injectable which can be injected, for example, via a syringe or aninfusion pump. The injection can be delivered subcutaneously,intramuscularly, intraperitoneally, or intravenously, for example.

In another embodiment, the pharmaceutical composition is a solidformulation, e.g., a freeze-dried or spray-dried composition, which canbe used as is, or whereto the physician or the patient adds solvents,and/or diluents prior to use. Solid dosage forms can include tablets,such as compressed tablets, and/or coated tablets, and capsules (e.g.,hard or soft gelatin capsules). The pharmaceutical composition can alsobe in the form of sachets, dragees, powders, granules, lozenges, orpowders for reconstitution, for example.

The dosage forms can be immediate release, in which case they cancomprise a water-soluble or dispersible carrier, or they may be delayedrelease, sustained release, or modified release, in which case they maycomprise water-insoluble polymers that regulate the rate of dissolutionof the dosage form in the gastrointestinal tract.

In other embodiments, the pharmaceutical composition can be deliveredintranasally, intrabuccally, or sublingually.

The pH in an aqueous formulation can be between pH 3 and pH 10. In oneembodiment of the invention, the pH of the formulation is from about 7.0to about 9.5. In another embodiment of the invention, the pH of theformulation is from about 3.0 to about 7.0.

In another embodiment of the invention, the pharmaceutical compositioncomprises a buffer. Non-limiting examples of buffers include: arginine,aspartic acid, bicine, citrate, disodium hydrogen phosphate, fumaricacid, glycine, glycylglycine, histidine, lysine, maleic acid, malicacid, sodium acetate, sodium carbonate, sodium dihydrogen phosphate,sodium phosphate, succinate, tartaric acid, tricine, andtris(hydroxymethyl)-aminomethane, and mixtures thereof. The buffer maybe present individually or in the aggregate, in a concentration fromabout 0.01 mg/ml to about 50 mg/ml, for example from about 0.1 mg/ml toabout 20 mg/ml. Pharmaceutical compositions comprising each one of thesespecific buffers constitute alternative embodiments of the invention.

In another embodiment of the invention, the pharmaceutical compositioncomprises a preservative. Non-limiting examples of preservativesinclude: benzethonium chloride, benzoic acid, benzyl alcohol, bronopol,butyl 4-hydroxybenzoate, chlorobutanol, chlorocresol, chlorohexidine,chlorphenesin, o-cresol, m-cresol, p-cresol, ethyl 4-hydroxybenzoate,imidurea, methyl 4-hydroxybenzoate, phenol, 2-phenoxyethanol,2-phenylethanol, propyl 4-hydroxybenzoate, sodium dehydroacetate,thiomerosal, and mixtures thereof. The preservative may be presentindividually or in the aggregate, in a concentration from about 0.01mg/ml to about 50 mg/ml, for example from about 0.1 mg/ml to about 20mg/ml. Pharmaceutical compositions comprising each one of these specificpreservatives constitute alternative embodiments of the invention.

In another embodiment of the invention, the pharmaceutical compositioncomprises an isotonic agent. Non-limiting examples of isotonic agentsinclude a salt (such as sodium chloride), an amino acid (such asglycine, histidine, arginine, lysine, isoleucine, aspartic acid,tryptophan, and threonine), an alditol (such as glycerol,1,2-propanediol propyleneglycol), 1,3-propanediol, and 1,3-butanediol),polyethyleneglycol (e.g. PEG400), and mixtures thereof. Another exampleof an isotonic agent includes a sugar. Non-limiting examples of sugarsmay be mono-, di-, or polysaccharides, or water-soluble glucans,including for example fructose, glucose, mannose, sorbose, xylose,maltose, lactose, sucrose, trehalose, dextran, pullulan, dextrin,cyclodextrin, alpha and beta-HPCD, soluble starch, hydroxyethyl starch,and sodium carboxymethylcellulose. Another example of an isotonic agentis a sugar alcohol, wherein the term “sugar alcohol” is defined as aC(4-8) hydrocarbon having at least one —OH group. Non-limiting examplesof sugar alcohols include mannitol, sorbitol, inositol, galactitol,dulcitol, xylitol, and arabitol. Pharmaceutical compositions comprisingeach isotonic agent listed in this paragraph constitute alternativeembodiments of the invention. The isotonic agent can be presentindividually or in the aggregate, in a concentration from about 0.01mg/ml to about 50 mg/ml, for example from about 0.1 mg/ml to about 20mg/ml. Pharmaceutical compositions comprising each one of these specificisotonic agents constitute alternative embodiments of the invention.

In another embodiment of the invention, the pharmaceutical compositioncomprises a chelating agent. Non-limiting examples of chelating agentsinclude citric acid, aspartic acid, salts of ethylenediaminetetraaceticacid (EDTA), and mixtures thereof. The chelating agent can be presentindividually or in the aggregate, in a concentration from about 0.01mg/ml to about 50 mg/ml, for example from about 0.1 mg/ml to about 20mg/ml. Pharmaceutical compositions comprising each one of these specificchelating agents constitute alternative embodiments of the invention.

In another embodiment of the invention, the pharmaceutical compositioncomprises a stabilizer. Non-limiting examples of stabilizers include oneor more aggregation inhibitors, one or more oxidation inhibitors, one ormore surfactants, and/or one or more protease inhibitors.

In another embodiment of the invention, the pharmaceutical compositioncomprises a stabilizer, wherein said stabilizer iscarboxy-/hydroxycellulose and derivates thereof (such as HPC, HPC-SL,HPC-L and HPMC), cyclodextrins, 2-methylthioethanol, polyethylene glycol(such as PEG 3350), polyvinyl alcohol (PVA), polyvinyl pyrrolidone,salts (such as sodium chloride), sulphur-containing substances such asmonothioglycerol), or thioglycolic acid. The stabilizer can be presentindividually or in the aggregate, in a concentration from about 0.01mg/ml to about 50 mg/ml, for example from about 0.1 mg/ml to about 20mg/ml. Pharmaceutical compositions comprising each one of these specificstabilizers constitute alternative embodiments of the invention.

In further embodiments of the invention, the pharmaceutical compositioncomprises one or more surfactants, preferably a surfactant, at least onesurfactant, or two different surfactants. The term “surfactant” refersto any molecules or ions that are comprised of a water-soluble(hydrophilic) part, and a fat-soluble (lipophilic) part. The surfactantcan, for example, be selected from the group consisting of anionicsurfactants, cationic surfactants, nonionic surfactants, and/orzwitterionic surfactants. The surfactant can be present individually orin the aggregate, in a concentration from about 0.1 mg/ml to about 20mg/ml. Pharmaceutical compositions comprising each one of these specificsurfactants constitute alternative embodiments of the invention.

In a further embodiment of the invention, the pharmaceutical compositioncomprises one or more protease inhibitors, such as, e.g., EDTA(ethylenediamine tetraacetic acid), and/or benzamidine hydrochloric acid(HCl). The protease inhibitor can be present individually or in theaggregate, in a concentration from about 0.1 mg/ml to about 20 mg/ml.Pharmaceutical compositions comprising each one of these specificprotease inhibitors constitute alternative embodiments of the invention.

The pharmaceutical composition of the invention can comprise an amountof an amino acid base sufficient to decrease aggregate formation of thepolypeptide during storage of the composition. The term “amino acidbase” refers to one or more amino acids (such as methionine, histidine,imidazole, arginine, lysine, isoleucine, aspartic acid, tryptophan,threonine), or analogues thereof. Any amino acid can be present eitherin its free base form or in its salt form.

Any stereoisomer (i.e., L, D, or a mixture thereof) of the amino acidbase may be present. The amino acid base can be present individually orin the combination with other amino acid bases, in a concentration fromabout 0.01 mg/ml to about 50 mg/ml, for example from about 0.1 mg/ml toabout 20 mg/ml. Pharmaceutical compositions comprising each one of thesespecific amino acid bases constitute alternative embodiments of theinvention.

It is also apparent to one skilled in the art that the therapeuticallyeffective dose for adenoviral particles comprising a nucleic acidmolecule encoding a Zika virus antigen of the present invention or apharmaceutical composition thereof will vary according to the desiredeffect. Therefore, optimal dosages to be administered can be readilydetermined by one skilled in the art and will vary with the particularadenoviral particle used, the mode of administration, the strength ofthe preparation, and the advancement of the disease condition (e.g.,Zika virus infection). In addition, factors associated with theparticular subject being treated, including subject age, weight, dietand time of administration, will result in the need to adjust the doseto an appropriate therapeutic level.

The pharmaceutically-acceptable salts of the adenoviral particles of theinvention include the conventional non-toxic salts or the quaternaryammonium salts which are formed from inorganic or organic acids orbases. Examples of such acid addition salts include acetate, adipate,benzoate, benzenesulfonate, citrate, camphorate, dodecylsulfate,hydrochloride, hydrobromide, lactate, maleate, methanesulfonate,nitrate, oxalate, pivalate, propionate, succinate, sulfate and tartrate.Base salts include ammonium salts, alkali metal salts such as sodium andpotassium salts, alkaline earth metal salts such as calcium andmagnesium salts, salts with organic bases such as dicyclohexylaminosalts and salts with amino acids such as arginine. Also, the basicnitrogen-containing groups may be quaternized with, for example, alkylhalides.

The pharmaceutical compositions of the invention can be administered byany means that accomplish their intended purpose. As used herein, by“administering” is meant a method of giving a dosage of a pharmaceuticalcomposition (e.g., an immunogenic composition (e.g., a vaccine (e.g., aZika virus (ZIKV) vaccine))) to a subject. The compositions utilized inthe methods described herein can be administered, for example,intramuscularly, intravenously, intradermally, percutaneously,intraarterially, intraperitoneally, intralesionally, intracranially,intraarticularly, intraprostatically, intrapleurally, intratracheally,intranasally, intravitreally, intravaginally, intrarectally, topically,intratumorally, peritoneally, subcutaneously, subconjunctivally,intravesicularlly, mucosally, intrapericardially, intraumbilically,intraocularly, orally, topically, locally, by inhalation, by injection,by infusion, by continuous infusion, by localized perfusion bathingtarget cells directly, by catheter, by lavage, by gavage, in cremes, orin lipid compositions. The preferred method of administration can varydepending on various factors (e.g., the components of the compositionbeing administered and the severity of the condition being treated).

Methods of Use

The present invention provides methods for generating an immune responseagainst a Zika virus in a human subject in need thereof. The methodscomprise administering to the subject a pharmaceutical compositioncomprising adenoviral vectors comprising a nucleic acid sequenceencoding a Zika virus antigen and a pharmaceutically acceptable carrier.The methods are for preventing, treating, delaying the onset of, orameliorating a Zika virus infection or any one or more symptoms of saidZika virus infection, the method comprising administering to the subjectin need thereof an effective amount of a pharmaceutical composition ofthe invention.

According to particular embodiments, an immunogenic or effective orprotective amount refers to the amount of an immunogen which issufficient to achieve one, two, three, four, or more of the followingeffects: (i) reduce or ameliorate the severity of the Zika virusinfection to be treated or a symptom associated therewith; (ii) reducethe duration of the Zika virus infection to be treated, or a symptomassociated therewith; (iii) prevent the progression of the Zika virusinfection to be treated, or a symptom associated therewith; (iv) causeregression of the Zika virus infection to be treated, or a symptomassociated therewith; (v) prevent the development or onset of the Zikavirus infection to be treated, or a symptom associated therewith; (vi)prevent the recurrence of the Zika virus infection to be treated, or asymptom associated therewith; (vii) reduce hospitalization of a subjecthaving the Zika virus infection or condition to be treated, or a symptomassociated therewith; (viii) reduce hospitalization length of a subjecthaving the Zika virus infection to be treated, or a symptom associatedtherewith; (ix) increase the survival of a subject with the Zika virusinfection to be treated, or a symptom associated therewith; (xi) inhibitor reduce the Zika virus infection to be treated, or a symptomassociated therewith in a subject; and/or (xii) enhance or improve theprophylactic or therapeutic effect(s) of another therapy; (xiii) preventtransmission of zika virus through sexual and maternal to fetal routes;(xiv) prevent and/or reduces the severity of fetal brain abnormalitiesassociated with Zika virus.

Examples of symptoms of diseases caused by a viral infection, such asZIKV, that can be prevented using the compositions of the inventioninclude, for example, fever, joint pain, rash, conjunctivitis, musclepain, headache, retro-orbital pain, edema, lymphadenopathy, malaise,asthenia, sore throat, cough, nausea, vomiting, diarrhea, andhematospermia. These symptoms, and their resolution during treatment,can be measured by, for example, a physician during a physicalexamination or by other tests and methods known in the art.

The immunogenic or effective amount or dosage can vary according tovarious factors, such as the Zika virus infection to be treated, themeans of administration, the target site, the physiological state of thesubject (including, e.g., age, body weight, health), whether the subjectis a human or an animal, other medications administered, and whether thetreatment is prophylactic or therapeutic. Treatment dosages areoptimally titrated to optimize safety and efficacy.

As used herein, the terms “treat,” “treating,” and “treatment” are allintended to refer to an amelioration or reversal of at least onemeasurable physical parameter related to the Zika virus infection, whichis not necessarily discernible in the subject, but can be discernible inthe subject. The terms “treat,” “treating,” and “treatment,” can alsorefer to causing regression, preventing the progression, or at leastslowing down the progression of the Zika virus infection. In aparticular embodiment, “treat,” “treating,” and “treatment” refer to analleviation, prevention of the development or onset, or reduction in theduration of one or more symptoms associated with the Zika virusinfection. In a particular embodiment, “treat,” “treating,” and“treatment” refer to prevention of the recurrence of the Zika virusinfection. In a particular embodiment, “treat,” “treating,” and“treatment” refer to an increase in the survival of a subject having theZika virus infection. In a particular embodiment, “treat,” “treating,”and “treatment” refer to elimination of the Zika virus infection in thesubject.

In certain embodiments, administration of an immunogenic or effectiveamount of a pharmaceutical composition of the invention reduces ZIKVserum viral loads determined from a subject having a ZIKV infection byat least about 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or morecompared to viral loads determined from the subject prior toadministration of an effective amount of a composition of the invention.In some instances, administration of an effective amount of acomposition of the invention reduces serum viral loads to anundetectable level compared to viral loads determined from the subjectprior to administration of an effective amount of a composition of theinvention. In some instances, administration of an effective amount of acomposition of the invention results in a reduced and/or undetectableserum viral load that may be maintained for at least about 1, 2, 3, 4,5, 6, 7 days; 1, 2, 3, 4, weeks; 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or12 months; or 1 year or more.

In addition, single or multiple administrations of the compositions ofthe present invention can be given (pre- or post-exposure and/or pre- orpost-diagnosis) to a subject (e.g., one administration or administrationtwo or more times). For example, subjects who are particularlysusceptible to, for example, viral infection (e.g., a ZIKV infection)can require multiple administrations of the compositions of the presentinvention to establish and/or maintain protection against the virus.Levels of induced immunity provided by the pharmaceutical compositionsdescribed herein can be monitored by, for example, measuring amounts ofneutralizing secretory and serum antibodies. The dosages can then beadjusted or repeated as necessary to trigger the desired level of immuneresponse. For example, the immune response triggered by a singleadministration (prime) of a composition of the invention may notsufficiently potent and/or persistent to provide effective protection.Accordingly, in some embodiments, repeated administration (boost), suchthat a prime boost regimen is established, can significantly enhancehumoral and cellular responses to the antigen of the composition.

Alternatively, the efficacy of treatment can be determined by monitoringthe level of the antigenic or therapeutic gene product, or fragmentthereof, expressed in a subject (e.g., a human) following administrationof the pharmaceutical compositions of the invention. For example, theblood or lymph of a subject can be tested for antigenic or therapeuticgene product, or fragment thereof, using, for example, standard assaysknown in the art.

In some instances, efficacy of treatment can be determined by monitoringa change in the serum viral load from a sample from the subject obtainedprior to and after administration of an effective amount of apharmaceutical composition of the invention. A reduction in serum viralload of at least about 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%,or more compared to viral load determined from the subject prior toadministration of an effective amount of a composition of the inventioncan indicate that the subject is receiving benefit from the treatment.If a viral load does not decrease by at least about 10%, 20%, 30%, ormore after administration of a composition of the invention, the dosageof the composition to be administered can be increased. For example, byincreasing the number of viral particles (VP) of an adenovirusvector-based vaccine.

Immunogenicity of the pharmaceutical compositions of the invention canbe improved if it is co-administered with an immunostimulatory agentand/or adjuvant. Suitable adjuvants well-known to those skilled in theart include, for example, aluminum phosphate, aluminum hydroxide, QS21,Quil A (and derivatives and components thereof), calcium phosphate,calcium hydroxide, zinc hydroxide, glycolipid analogs, octodecyl estersof an amino acid, muramyl dipeptides, polyphosphazene, lipoproteins,ISCOM matrix, DC-Chol, DDA, cytokines, and other adjuvants andderivatives thereof.

The term “immunostimulatory agent” refers to substances (e.g., drugs andnutrients) that stimulate the immune system by inducing activation orincreasing activity of any of its components. An immunostimulatory agentcan, for example, include a cytokine (e.g., the granulocyte macrophagecolony-stimulating factor) and interferon (e.g., IFN-α and/or IFN-γ).

The term “adjuvant” is defined as a pharmacological or immunologicalagent that modifies the effect of other agents (e.g., a ZIKV antigen)while having few if any direct effects when administered alone. Anadjuvant can be one or more substances that cause stimulation of theimmune system. In this context, an adjuvant is used to enhance an immuneresponse to the adenoviral particles of the invention.

EMBODIMENTS

The invention provides also the following non-limiting embodiments.

Embodiment 1 is a method for generating an immune response against aZika virus in a human subject in need thereof, the method comprisingadministering to the subject a pharmaceutical composition comprisingadenoviral vectors comprising a nucleic acid sequence encoding a Zikavirus antigen and a pharmaceutically acceptable carrier, wherein about5×10¹⁰ adenoviral vectors (or particles) to about 1×10¹¹ adenoviralvectors (or particles) are administered per dose to the subject in needthereof.

Embodiment 2 is the method of embodiment 1, for generating a protectiveimmune response against a Zika virus in a human subject in need thereof.

Embodiment 3 is the method of embodiment 1 or 2, wherein thepharmaceutical composition is administered via an intramuscularinjection to the human subject in need thereof.

Embodiment 4 is the method of any one of embodiments 1-3, wherein thepharmaceutical composition is administered to the human subject as asingle dose.

Embodiment 5 is the method of any one of embodiments 1-3, wherein thepharmaceutical composition is administered to the human subject as adouble dose.

Embodiment 6 is the method of embodiment 5, wherein the first and seconddose of the pharmaceutical composition are administered to the humansubject about four weeks, about eight weeks, about twelve weeks, aboutthree months, about six months, about nine months, about one year, orabout two years apart.

Embodiment 7 is the method of embodiment 5, wherein the first and seconddose of the pharmaceutical composition are administered to the humansubject about eight weeks apart.

Embodiment 8 is the method of any one of embodiments 1-7, wherein about5×10¹⁰, about 6×10¹⁰, about 7×10¹⁰, about 8×10¹⁰, about 9×10¹⁰, or about1×10¹¹ of the adenoviral vectors (or particles) are administered to thehuman subject in need thereof.

Embodiment 9 is the method of embodiment 8, wherein about 5×10¹⁰adenoviral vectors (or particles) are administered to the human subjectin need thereof.

Embodiment 10 is the method of embodiment 8, wherein about 1×10¹¹adenoviral vectors (or particles) are administered to the human subjectin need thereof.

Embodiment 11 is the method of any one of embodiments 1-10, wherein theZika virus antigen comprises the amino acid sequence of SEQ ID NO:2, 4,6, 8, 10, or 12.

Embodiment 12 is the method of embodiment 11, wherein the Zika virusantigen comprises the amino acid sequence of SEQ ID NO:2.

Embodiment 13 is the method of embodiment 11, wherein the Zika virusantigen comprises the amino acid sequence of SEQ ID NO:4.

Embodiment 14 is the method of embodiment 11, wherein the Zika virusantigen comprises the amino acid sequence of SEQ ID NO:6.

Embodiment 15 is the method of embodiment 11, wherein the Zika virusantigen comprises the amino acid sequence of SEQ ID NO:8.

Embodiment 16 is the method of embodiment 11, wherein the Zika virusantigen comprises the amino acid sequence of SEQ ID NO:10.

Embodiment 17 is the method of embodiment 11, wherein the Zika virusantigen comprises the amino acid sequence of SEQ ID NO:12.

Embodiment 18 is the method of any one of embodiments 1-17, wherein theadenoviral vector serotype is selected from the group consisting of Ad2,Ad5, Ad11, Ad12, Ad24, Ad26, Ad34, Ad35, Ad40, Ad48, Ad49, Ad50, Ad52,and Pan9.

Embodiment 19 is the method of embodiment 18, wherein the adenoviralvector serotype is the Ad26 serotype.

Embodiment 20 is a method for preventing a Zika virus infection or theprogression of a Zika virus infection in a human subject in needthereof, the method comprising administering to the subject apharmaceutical composition comprising adenoviral vectors comprising anucleic acid sequence encoding a Zika virus antigen and apharmaceutically acceptable carrier, wherein the pharmaceuticalcomposition comprises a total dosage of about 5×10¹⁰ adenoviral vectors(or particles) to about 1×10¹¹ adenoviral vectors (or particles) peradministration.

Embodiment 21 is the method of embodiment 20, wherein the pharmaceuticalcomposition is administered intramuscularly, intravenously,intradermally, percutaneously, intraarterially, intraperitoneally,intralesionally, intracranially, intraarticularly, intraprostatically,intrapleurally, intratracheally, intranasally, intravitreally,intravaginally, intrarectally, topically, intratumorally, peritoneally,subcutaneously, subconjunctivally, intravesicularlly, mucosally,intrapericardially, intraumbilically, intraocularly, orally, topically,locally, by inhalation, by injection, by infusion, by continuousinfusion, by localized perfusion, by catheter, by lavage, or by gavage.

Embodiment 22 is the method of embodiment 21, wherein the pharmaceuticalcomposition is administered in an intramuscular injection.

Embodiment 23 is the method of any one of embodiments 20-22, wherein thepharmaceutical composition is administered as a single dose.

Embodiment 24 is the method of any one of embodiments 20-22, wherein thepharmaceutical composition is administered as a double dose.

Embodiment 25 is the method of embodiment 24, wherein the first andsecond dose of the pharmaceutical composition are administered to thehuman subject about four weeks, about eight weeks, about twelve weeks,about three months, about six months, about nine months, about one year,or about two years apart.

Embodiment 26 is the method of embodiment 25, wherein the first andsecond dose of the pharmaceutical composition are administered to thehuman subject about eight weeks apart.

Embodiment 27 is the method of any one of the embodiments 20-26, whereinabout 5×10¹⁰, about 6×10¹⁰, about 7×10¹⁰, about 8×10¹⁰, about 9×10¹⁰, orabout 1×10¹¹ adenoviral vectors (or particles) are administered to thehuman subject in need thereof.

Embodiment 28 is the method of embodiment 27, wherein about 5×10¹⁰adenoviral vectors (or particles) are administered to the human subjectin need thereof.

Embodiment 29 is the method of embodiment 27, wherein about 1×10¹¹adenoviral vectors (or particles) are administered to the human subjectin need thereof.

Embodiment 30 is the method of any one of embodiments 20-29, wherein theZika virus antigen comprises the amino acid sequence of SEQ ID NO:2, 4,6, 8, 10, or 12.

Embodiment 31 is the method of embodiment 30, wherein the Zika virusantigen comprises the amino acid sequence of SEQ ID NO:2.

Embodiment 32 is the method of embodiment 30, wherein the Zika virusantigen comprises the amino acid sequence of SEQ ID NO:4.

Embodiment 33 is the method of embodiment 30, wherein the Zika virusantigen comprises the amino acid sequence of SEQ ID NO:6.

Embodiment 34 is the method of embodiment 30, wherein the Zika virusantigen comprises the amino acid sequence of SEQ ID NO:8.

Embodiment 35 is the method of embodiment 30, wherein the Zika virusantigen comprises the amino acid sequence of SEQ ID NO:10.

Embodiment 36 is the method of embodiment 30, wherein the Zika virusantigen comprises the amino acid sequence of SEQ ID NO:12.

Embodiment 37 is the method of any one of embodiments 20-36, wherein theadenoviral vector serotype is selected from the group consisting of Ad2,Ad5, Ad11, Ad12, Ad24, Ad26, Ad34, Ad35, Ad40, Ad48, Ad49, Ad50, Ad52,and Pan9.

Embodiment 38 is the method of embodiment 37, wherein the adenoviralvector serotype is the Ad26 serotype.

Embodiment 39 is the use of a pharmaceutical composition for thepreparation of a vaccine for generating an immune response against aZika virus in a human subject in need thereof, comprising adenoviralvectors comprising a nucleic acid sequence encoding a Zika virus antigenand a pharmaceutically acceptable carrier, wherein about 5×10¹⁰adenoviral vectors (or particles) to about 1×10¹¹ adenoviral vectors (orparticles) are administered per dose to the subject in need thereof.

Embodiment 40 is the use of embodiment 39, wherein the vaccine isadministered intramuscularly, intravenously, intradermally,percutaneously, intraarterially, intraperitoneally, intralesionally,intracranially, intraarticularly, intraprostatically, intrapleurally,intratracheally, intranasally, intravitreally, intravaginally,intrarectally, topically, intratumorally, peritoneally, subcutaneously,subconjunctivally, intravesicularlly, mucosally, intrapericardially,intraumbilically, intraocularly, orally, topically, locally, byinhalation, by injection, by infusion, by continuous infusion, bylocalized perfusion, by catheter, by lavage, or by gavage.

Embodiment 41 is the use of embodiment 40, wherein the vaccine isadministered in an intramuscular injection.

Embodiment 42 is the use of any one of embodiments 39-41, wherein thevaccine is administered as a single dose.

Embodiment 43 is the use of any one of embodiments 39-41, wherein thevaccine is administered as a double dose.

Embodiment 44 is the use of embodiment 43, wherein the first and seconddose of the vaccine are administered to the human subject about fourweeks, about eight weeks, about twelve weeks, about three months, aboutsix months, about nine months, about one year, or about two years apart.

Embodiment 45 is the use of embodiment 44, wherein the first and seconddose of the vaccine are administered to the human subject about eightweeks apart.

Embodiment 46 is the use of any one of the embodiments 39-45, whereinabout 5×10¹⁰, about 6×10¹⁰, about 7×10¹⁰, about 8×10¹⁰, about 9×10¹⁰, orabout 1×10¹¹ adenoviral vectors (or particles) are administered to thehuman subject in need thereof.

Embodiment 47 is the use of embodiment 46, wherein about 5×10¹⁰adenoviral vectors (or particles) are administered to the human subjectin need thereof.

Embodiment 48 is the use of embodiment 46, wherein about 1×10¹¹adenoviral vectors (or particles) are administered to the human subjectin need thereof.

Embodiment 49 is the use of any one of embodiments 39-48, wherein theZika virus antigen comprises the amino acid sequence of SEQ ID NO:2, 4,6, 8, 10, or 12.

Embodiment 50 is the use of embodiment 49, wherein the Zika virusantigen comprises the amino acid sequence of SEQ ID NO:2.

Embodiment 51 is the use of embodiment 49, wherein the Zika virusantigen comprises the amino acid sequence of SEQ ID NO:4.

Embodiment 52 is the use of embodiment 49, wherein the Zika virusantigen comprises the amino acid sequence of SEQ ID NO:6.

Embodiment 53 is the use of embodiment 49, wherein the Zika virusantigen comprises the amino acid sequence of SEQ ID NO:8.

Embodiment 54 is the use of embodiment 49, wherein the Zika virusantigen comprises the amino acid sequence of SEQ ID NO:10.

Embodiment 55 is the use of embodiment 49, wherein the Zika virusantigen comprises the amino acid sequence of SEQ ID NO:12.

Embodiment 56 is the use of any one of embodiments 39-55, wherein theadenoviral vector serotype is selected from the group consisting of Ad2,Ad5, Ad11, Ad12, Ad24, Ad26, Ad34, Ad35, Ad40, Ad48, Ad49, Ad50, Ad52,and Pan9.

Embodiment 57 is the use of embodiment 56, wherein the adenoviral vectorserotype is the Ad26 serotype.

EXAMPLES Example 1: Clinical Study to Evaluate Safety, Tolerability, andImmunogenicity of Intramuscularly Administered Adenoviral ParticlesComprising a Zika Virus Antigen

A clinical study was conducted for evaluating the safety, tolerabilityand immunogenicity of Ad26.ZIKV.001 vectors at a dose of 5×10¹⁰ viralparticles (vp) or 1×10¹¹ vp, which were administered intramuscularly assingle dose or in a two dose schedule in healthy adults.

The study was a randomized, placebo-controlled, double-blind first inhuman phase 1 study conducted in 100 healthy adult male and femalesubjects (>18 to <50 years of age). The study consists of a vaccinationperiod in which subjects were administered the Ad26.ZIKV.001 vaccine ateither a 5×10¹⁰ vp or 1×10¹¹ vp dose at baseline (Day 1) followed by asecond administration on Day 57 of either a placebo or the Ad26.ZIKV.001vaccine at a similar dose as the first administration.

Subjects were enrolled into 5 different groups of 20 healthy subjectseach. Overall, subjects were randomized in parallel in 1:1:1:1:1 ratioto 1 of 5 vaccination groups to receive active vaccine or placebo (0.9%saline) through intramuscular (IM) injections (0.5 ml) as follows:

Group 1: Ad26.ZIKV.001 (5×10¹⁰ vp) on Day 1, followed by a secondadministration of Ad26.ZIKV.001 (5×10¹⁰ vp) on Day 57, or

Group 2: Ad26.ZIKV.001 (5×10¹⁰ vp) on Day 1, followed by a Placebo onDay 57.

Group 3: Ad26.ZIKV.001 (1×10¹¹ vp) on Day 1, followed by a secondadministration of Ad26.ZIKV.001 (1×10¹¹ vp) on Day 57, or

Group 4: Ad26.ZIKV.001 (1×10¹¹ vp) on Day 1, followed by a Placebo onDay 57.

Group 5: Placebo on Day 1, followed by a Placebo on Day 57.

The exemplary study vaccination schedules are summarized in Table 1.

TABLE 1 Study Vaccination Schedules Immunization Group Immunization 1Immunization 2 Schedule Size Group (Dose 1) (Dose 2) (Weeks) N 1Ad26.ZIKV.001 Ad26.ZIKV.001 0-8 20 (5 × 10¹⁰ vp) (5 × 10¹⁰ vp) 2Ad26.ZIKV.001 Placebo 0-8 20 (5 × 10¹⁰ vp) 3 Ad26.ZIKV.001 Ad26.ZIKV.0010-8 20 (1 × 10¹¹ vp) (1 × 10¹¹ vp) 4 Ad26.ZIKV.001 Placebo 0-8 20 (1 ×10¹¹ vp) . 5 Placebo Placebo 0-8 20 Abbreviations: vp: viral particles;N: number of subjects to receive study vaccine

Vaccine Material

Ad26.ZIKV.001 is a monovalent vaccine composed of a recombinant,replication-incompetent adenovirus serotype 26 (Ad26) vector,constructed to encode the Zika virus (ZIKV) membrane (M) and envelope(Env) proteins (M-Env) (SEQ ID NO:2) derived from the ZIKV strain fromBrazil (ZIKV-BR) BeH815744 strain.

Safety Endpoint

Safety was assessed by a collection of solicited local and systemicadverse events, unsolicited adverse events, serious adverse events, andimmediately reportable events. Other safety assessments includedclinical laboratory tests (hematology, biochemistry), vital signsmeasurements (heart rate, supine systolic and diastolic blood pressure,body temperature), and physical examinations at multiple time points.

Immunogenicity Endpoints

Blood samples for immunogenicity assessments (humoral and cellularimmune responses) were collected at different time points. Blood samples(serum) for assessment of humoral immune responses were obtained fromall subjects. Blood samples (PBMC) for assessment of cellular immuneresponses were obtained from a subset of subjects only.

Humoral and cellular immunogenicity assays can include, but are notlimited to, the assays summarized in Tables 2 and 3.

TABLE 2 Summary of Humoral Immunogenicity Assays Assay Purpose Secondaryendpoints ZIKV neutralization Analysis of neutralizing antibodies to the(VNA) vaccine strain (or other strain) Exploratory endpoints ZIKVantibody Analysis of antibodies binding to ZIKV, or (ELISA) individualZIKV proteins (e.g., Env-protein, M-protein) ZIKV neutralizationAnalysis of neutralizing antibodies to vaccine (neutralization strain(or other strain), as measured by an assay) alternative neutralizationassay (different from the VNA used for the secondary endpoint)Flavivirus Analysis of neutralizing antibodies to neutralization (VNA)flaviviruses other than ZIKV, such as YFV, JEV, WNV, DENV, and TBEVELISA: enzyme-linked immunosorbent assay

TABLE 3 Summary of Cellular Immunogenicity Assays Assay Exploratoryendpoints Purpose Flow cytometry (ICS) Analysis of T-cell responses toZIKV M-, Env-, and/or other protein peptides (including, but not limitedto CD4+/CD8+, IL2, IFNγ, TNFα, other markers determining functionality,memory differentiation, activation or T-helper [Th]1/Th2 status) IFNγELISpot T-cell IFNγ responses to ZIKV M-, Env-, and/or other ZIKVprotein peptides, or adenoviral protein peptide ELISpot: enzyme-linkedimmunspot; ICS: intracellular cytokine staining; IFN: interferon; IL:interleukin; TNF: tumor necrosis factor

Immunogenicity was assessed in a subset of 75 subjects at baseline (Day1), at 14 days and 28 days after the first vaccination (Days 15 and 29),at the day of the boost vaccination (pre-dose, D57), and 14 days and 28days after the boost vaccination (D71 and 85) using a virusneutralization assay (VNA) to analyze the neutralizing antibody responseagainst ZIKV (Zika Microneutralization Assay (MN50)). This in vitroassay determines the capacity of vaccine-induced antibodies to preventinfection of a cell line by live ZIKV (Modjarrad et al., The Lancet,Vol. 391, issue 10120, 10-16 Feb. 2018, p. 563-571). Results from theremaining 25 subjects, from any other immunological assessment measuringresponse against ZIKV, or from further time points in the ongoing studywere not yet available.

The actual values, geometric mean titers (GMT) and percentage ofresponders are shown in FIGS. 1A and B (Zika Microneutralization Assay(MN50). Table 4 shows the GMTs, the percentage of subjects with MN50titers above 10 (lower limit of quantification of the MN50 VNA and assaypositivity criterium) and 100 MN50 units, respectively, and thegeometric fold increase for the Zika microneutralization assay. Titersabove 100 MN50 units have been associated with protection against ZIKVchallenge in mouse and NHP models (Modjarrad et al., The Lancet, Vol.391, issue 10120, 10-16 Feb. 2018, p. 563-571; Abbink et al., Science, 9Sep. 2016, Vol. 353, Issue 6304, pp. 1129-1132; Abbink et al., SciTransl Med. 2017, December 13, 9(420)).

At baseline all subjects had a geometric mean titer below LLOQ. None ofthe placebo recipients developed Zika MN50 titers above the LLOQ of 10at any time point post vaccination. Fifteen days post dose 1, a majorityof vaccinated subjects developed ZIKV neutralizing antibody titers,irrespective of the dose given, and geometric mean titers increased tolevels above 100 MN50 units in all groups by day 28. Specifically,geometric mean titers at Day 15 after the first vaccination were 58.8and 29.8 respectively for the groups receiving Ad26.ZIKV.001 at 5×10¹⁰viral particles (vp), resulting in a seroconversion rate (i.e. subjectswith MN50 titers ≥10) of 80% (12/15) and 53% (8/15), and 53.5 and 52.9for the groups that received Ad26.ZIKV.001 at 1×10¹¹ vp, resulting in aseroconversion rate of 78.6% (11/14) and 66.7% (10/15), respectively.Geometric mean titers at Day 29 after the first vaccination increased to121.4 and 139.4 respectively for the groups receiving Ad26.ZIKV.001 at5×10¹⁰ vp, resulting in seroconversion rates of 93.3% (14/15) and 86.7%(13/15), and 125 and 169 for the groups that received Ad26.ZIKV.001 at1×10¹¹ vp, resulting in seroconversion rates of 92.2% (13/14) for bothgroups. The percentage of subjects whose MN50 titers exceeded 100—alevel that showed protection against ZIKV challenge in mouse and NHPmodels—increased from 36.7% (11/30) on day 15 to 56.7% (17/30) on day 28in the combined groups receiving 5×10¹⁰ vp of Ad26.ZIKV.001. In thecombined groups that received Ad26.ZIKV.001 at 1×10¹¹ vp, 13/29 (46.7%)and 16/28 (58.6%) subjects reached MN50 titers of >100 at 14 days and 28days after prime vaccination, respectively.

Geometric mean titers at Day 57, prior to the second dose of eithervaccine or placebo, were 37.5 and 64.9 respectively for the groupsreceiving 5×10¹⁰ vp of Ad26.ZIKV.001 and slightly higher, 127 and 120.3,for the groups that received 1×10¹¹ vp of Ad26.ZIKV.001 as prime. In thegroup that received placebo after prime with 5×10¹⁰ vp of Ad26.ZIKV.001,geometric mean titers remained at 64.2, 14 days after the secondvaccination (day 71) and decreased slightly further to 44.8, 28 daysafter the second vaccination (day 85).

In the group that received a boost of 5×10¹⁰ vp of Ad26.ZIKV.001 on day57, however, geometric mean MN50 titers increased almost 200-fold to1980.3 on day 71 and remained at 1284 on day 85, demonstrating a strongboost effect.

In the group that received placebo after prime with 1×10¹¹ vp ofAd26.ZIKV.001, geometric mean titers remained at 173.2 and 142.4 on day71 and 85, respectively, while they substantially increased to 1016.8and 1131 on days 71 and 85, respectively, in the group that received1×10¹¹ vp of Ad26.ZIKV.001 as boost, again demonstrating a strong boosteffect. All subjects receiving Ad26.ZIKV.001 at two doses, irrespectiveof the dose, developed MN50 titers ≥10 at both time points, resulting ina 100% seroconversion rate. In addition, 100% (14/14) in the group thatreceived two vaccinations of Ad26.ZIKV.001 at 5×10¹⁰ vp and 92.3%(12/13) in the group that received two vaccinations of Ad26.ZIKV.001 at1×10¹¹ vp developed MN50 titers ≥100—the threshold associated withprotection against ZIKV challenge in animal models—at 28 days after thesecond vaccination. In contrast, in the groups that received placeboafter the first vaccination of Ad26.ZIKV.001 at 5×10¹⁰ vp or at 1×10¹¹vp the percentage of subjects whose MN50 titers reached this thresholdwas 20% (3/15) and 61.5% (8/13) (at 28 days after the secondvaccination, respectively.

In summary, the results show that a robust humoral response was inducedwithin 4 weeks after priming with Ad26.ZIKV.001, and that a second doseof Ad26.ZIKV.001 administered eight weeks after priming was able toboost these responses to substantial levels.

TABLE 4 Zika Microneutralization Assay (MN50): Actual Values, FoldIncreases from Baseline and Percentage of Responders over Time; PerProtocol Immunogenicity Population (Study VAC26911ZIK1001) Ad26.ZIKV.001Ad26.ZIKV.001 Ad26.ZIKV.001 Ad26.ZIKV.001 ZIKV MN50 titer LD/LD LD/PLHD/HD HD/PL Placebo Baseline N 15 15 14 15 14  Geometric mean 5 (5; 5) 5(5; 5) 5 (5; 5) 5 (5; 5) 5 (5; 5) (95% CI) MN50 >= 10 n (%) 0 0 0 0 0MN50 >= 100 n (%) 0 0 0 0 0 Day 15 N 15 15 14 15 14  Geometric mean 58.8(22.5; 153.7) 29.8 (9.6; 92.6) 53.5 (17.1; 167.2) 52.9 (17.9; 156.2) 5(5; 5) (95% CI) Geometric mean fold 6.8 4.1 6.2 6.7 1 increaseResponders n (%) 12 (80.0%) 8 (53.3%) 11 (78.6%) 10 (66.7%) 0 MN50 >= 10n (%) 12 (80.0%) 8 (53.3%) 11 (78.6%) 10 (66.7%) 0 MN50 >= 100 n (%) 7(46.7%) 4 (26.7%) 6 (42.9%) 7 (46.7%) 0 Day 29 N 15 15 14 14 13 Geometric mean 121.4 (46.6; 316.6) 139.4 (50.1; 388.3) 125 (53.4; 292.5)169 (63.4; 450.7) 5 (5; 5) (95% CI) Geometric mean fold 12.7 15.3 13.117.8 1 increase Responders n (%) 14 (93.3%) 13 (86.7%) 13 (92.9%) 13(92.9%) 0 MN50 >= 10 n (%) 14 (93.3%) 13 (86.7%) 13 (92.9%) 13 (92.9%) 0MN50 >= 100 n (%) 8 (53.3%) 9 (60.0%) 8 (57.1%) 8 (57.1%) 0 Day 57 N 1415 13 13 13  Geometric mean 37.5 (14.7; 95.8) 64.9 (32; 131.6) 127(49.1; 328.3) 120.3 (49.7; 290.9) 5 (5; 5) (95% CI) Geometric mean fold4.6 6.8 13.4 12 1 increase Responders n (%) 10 (71.4%) 14 (93.3%) 12(92.3%) 13 (100.0%) 0 MN50 >= 10 n (%) 10 (71.4%) 14 (93.3%) 12 (92.3%)13 (100.0%) 0 MN50 >= 100 n (%) 4 (28.6%) 5 (33.3%) 8 (61.5%) 8 (61.5%)0 Day 71 N 14 15 13 13 13  Geometric mean 1980.3 (1296; 3025.7) 64.2(32.9; 125.1) 1016.8 (531.4; 1945.6) 173.2 (72.3; 415.1) 5 (5; 5) (95%CI) Geometric mean fold 198 7 101.7 17.3 1 increase Responders n (%) 14(100.0%) 13 (86.7%) 13 (100.0%) 13 (100.0%) 0 MN50 >= 10 n (%) 14(100.0%) 13 (86.7%) 13 (100.0%) 13 (100.0%) 0 MN50 >= 100 n (%) 14(100.0%) 7 (46.7%) 12 (92.3%) 7 (53.8%) 0 Day 85 N 14 15 13 13 13 Geometric mean 1284 (818.6; 2014) 44.8 (24.6; 81.5) 1131 (561.8; 2276.6)142.4 (47.2; 429.7) 5 (5; 5) (95% CI) Geometric mean fold 128.4 4.9113.1 15 1 increase Responders n (%) 14 (100.0%) 13 (86.7%) 13 (100.0%)12 (92.3%) 0 MN50 >= 10 n (%) 14 (100.0%) 13 (86.7%) 13 (100.0%) 12(92.3%) 0 MN50 >= 100 n (%) 14 (100.0%) 3 (20.0%) 12 (92.3%) 8 (61.5%) 0N: number of subjects with data Responder: 1) if baseline < LLOQ, R >=LLOQ 2) if baseline >= LLOQ, R = 4-fold increase from baseline FoldIncrease: 1) if baseline < LLOQ, FI = value post-baseline/LLOQ 2) ifbaseline >= LLOQ, FI = Value post-baseline/Baseline Value Note:Ad26.ZIKV.001 LD: Ad26.ZIKV.001 5 × 10¹⁰ vp; Ad26.ZIKV.001 HD:Ad26.ZIKV.001 1 × 10¹¹ vp; PL: Placebo

It will be appreciated by those skilled in the art that changes could bemade to the embodiments described above without departing from the broadinventive concept thereof. It is understood, therefore, that thisinvention is not limited to the particular embodiments disclosed, but itis intended to cover modifications within the spirit and scope of thepresent invention as defined by the present description.

It is claimed:
 1. A method for generating an immune response against aZika virus in a human subject in need thereof, the method comprisingadministering to the subject a pharmaceutical composition comprisingadenoviral vectors comprising a nucleic acid sequence encoding a Zikavirus antigen, wherein the Zika virus antigen comprises the amino acidsequence of SEQ ID NO:2, and a pharmaceutically acceptable carrier,wherein about 1×10¹¹ adenoviral vectors are administered in a singledose to the human subject in need thereof.
 2. The method of claim 1,wherein the pharmaceutical composition is administered via anintramuscular injection to the subject in need thereof.
 3. The method ofclaim 1, wherein the immune response is a protective immune responseagainst a Zika virus.
 4. The method of claim 1, wherein the adenoviralvectors are of the Ad26 serotype.
 5. A method for preventing a Zikavirus infection or the progression of a Zika virus infection in a humansubject in need thereof, the method comprising administering to thesubject a pharmaceutical composition comprising adenoviral vectorscomprising a nucleic acid sequence encoding a Zika virus antigen,wherein the Zika virus antigen comprises the amino acid sequence of SEQID NO:2, and a pharmaceutically acceptable carrier, wherein about 1×10¹¹adenoviral vectors are administered in a single dose to the humansubject in need thereof.
 6. The method of claim 5, wherein thepharmaceutical composition is administered via an intramuscularinjection to the subject in need thereof.
 7. The method of claim 5,wherein the adenoviral vectors are of the Ad26 serotype.